The goal of this project is to develop and validate a high throughput screening (HTS) assay to identify specific inhibitors of hexokinase domain containing 1 (HKDC1), a fifth mammalian hexokinase, which we have identified. We have found that this highly active glucose-phosphorylating enzyme is present in human pancreatic islets and is substantially up-regulated in the islets of diabetic mice, relative to non-diabetic ones. Moreover, we have demonstrated that glucose-dependent insulin secretion is down-regulated in islet cells expressing exogenous HKDC1. These data lead us to hypothesize that HKDC1 exacerbates the negative impact of high glucose on pancreatic insulin secreting [unreadable]-cells (glucotoxicity). Blocking HKDC1 may thus slow the decline in [unreadable]-cell function that occurs due to elevated blood glucose in Type 2 diabetes. Our strategy involves developing an automated high throughput spectroscopic assay that will measure hexokinase activity by following conversion of ATP to ADP (Specific Aim #1). Because of the great similarity between the different members of the hexokinase family (76% amino acid sequence identity and similar domain structure between HKDC1 and HK1), especially in the putative ATP-binding domain, great care must be taken to ensure the specificity of the identified HKDC1 modulators. Therefore, we will conduct the assay in an ATP-rich environment to select against molecules that bind to that domain. Consequently, we will monitor generation of ADP, rather than ATP depletion, as a measure of enzyme activity. Positive hits will then be subjected to secondary screening (Specific Aim #2) to confirm specificity, rule out possible artifacts and verify the capability of these compounds to exert their protective effects within intact pancreatic islet cells. Specific inhibitors would be of great value in defining the role of what is likely to be a key regulatory enzyme in metabolism in the organism as a whole and would provide the first test of the potential of HKDC1 as a target for pharmacologic therapy to prevent the [unreadable]-cell decline observed in Type 2 diabetes.